Microbiology of Barrier Component Analogues of a Deep Geological Repository

Microbiology of Barrier Component Analogues of a Deep Geological Repository by Rachel Beaver A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Biology Waterloo, Ontario, Canada, 2020 ©Rachel Beaver 2020 Author’s Declaration This thesis consists of material all of which I authored or co-authored: see Statement of Contributions included in the thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Statement of Contributions Chapter 2 The Tsukinuno bentonite sampling was coordinated by Erik Kremmer (NWMO). The Opalinus core was received from Niels Burzan and Rizlan Bernier-Latmani (École Polytechnique Fédérale de Lausanne, Switzerland). The Northern Ontario crystalline rock core sampling was coordinated by Jeff Binns (Nuclear Waste Management Organization). Sian Ford (McMaster University) swabbed the outer layer of the Northern Ontario crystalline rock core and crushed the inner layer. Melody Vachon (University of Waterloo) assisted with the cultivation of anaerobic heterotrophs and SRB. iii Abstract Many countries are in the process of designing and implementing long-term storage solutions for used nuclear fuel. Like many of these countries, Canada is considering a deep geological repository (DGR) wherein used fuel bundles will be placed in copper-coated carbon steel used fuel containers encased in bentonite buffer boxes. Previously published research has simulated aspects of a DGR experimentally to identify DGR conditions that would prevent microbial activity. Although such microcosm-type experiments can observe microbial growth and activity over relatively limited time frames, a DGR will remain functional for at least a million years, and will be exposed to fluctuating environmental conditions. To address these temporal limitations, there is value to examining ancient natural barrier components that serve as analogues for future DGR components. My research explored the abundance, viability, and composition of microorganisms in ancient natural DGR barrier component analogues using a combination of cultivation- and DNA-based approaches. Samples were obtained from the Tsukinuno bentonite deposit (Japan) that formed in the late Miocene (~10 million years ago), the Opalinus clay formation (Switzerland) that formed approximately 174 million years ago, and Canadian shield crystalline rock from Northern Ontario. Given sample logistics and site-access considerations, most cultivation and molecular analyses focussed on the Tsukinuno bentonite clay samples, although the other site samples were included for comparison using a subset of techniques. Aerobic heterotroph abundances were relatively low for all Tsukinuno bentonite samples, ranging from 4.1 x 101 to 1.1 x 104 colony forming units per gram dry weight (CFU/gdw). Anaerobic heterotrophs were only detected in half of the Tsukinuno bentonite samples and ranged in abundance from 4.5 x 101 CFU/gdw to 9.9 x 102 CFU/gdw. Sulfate-reducing bacteria were only detected in one sample by cultivation, with a very low estimate of 5 MPN/gdw. Overall, culturable microbial abundances were lower than nucleic acid targets detected by quantitative PCR (Wilcoxon signed ranks test; p <0.05). A range of 3.2 x 101 to 7.2 x 107 (average of 5.7 x 106) 16S ribosomal RNA (16S rRNA) gene copies/gdw was iv detected in the Tsukinuno bentonite samples, and averages of 1.2 x 103 16S rRNA gene copies/gdw and 1.1 x 103 16S rRNA gene copies/gdw were measured for the Opalinus clay and Northern Ontario crystalline rock (NOCR) core samples, respectively. Because this research did not use cultivation-based approaches for Opalinus clay and NOCR samples, and PCR-based technique cannot distinguish DNA from cells and extracellular DNA, future research would be required to determine whether the detected DNA was associated with viable microorganisms. Analysis of 16S rRNA gene amplicons revealed that three of the Tsukinuno bentonite samples were dominated by putative aerobic heterotrophs and fermenting bacteria from the Actinobacteria phylum, whereas five of the Tsukinuno bentonite samples were dominated by sequences similar to those from acidophilic chemolithoautotrophs capable of sulfur reduction. The remaining two Tsukinuno bentonite samples had nucleic acid profiles below reliable detection limits, generating inconsistent replicate 16S rRNA gene profiles and probable contaminant sequences. The NOCR and Opalinus clay samples also yielded inconsistent replicate 16S rRNA gene profiles, implying undetectable nucleic acids in those rock samples and profiles indistinguishable from contaminant sequences. Total genomic DNA from Tsukinuno clay cultures was extracted and 16S rRNA genes were amplified and sequenced. Representatives of the phyla Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes were identified in the aerobic heterotroph cultures, and additional bacteria from the phyla Actinobacteria and Firmicutes were isolated in the cultures of anaerobic heterotrophs and fermenting bacteria. Only one nucleic acid sequence detected from a culture was also detected in its corresponding clay sample, suggesting that nucleic acids from culturable bacteria were relatively rare within the clay samples. Sequencing of DNA extracted from the sulfate reducing bacteria culture revealed that the taxon present in the culture was affiliated with the Desulfosporosinus genus, which was not detected in the corresponding clay sample. vAcknowledgements I would like to thank everyone who helped and supported me throughout the completion of this degree. First, I would like to thank my supervisor, Josh Neufeld, for his continued support and enthusiasm, and for providing the resources necessary to complete this research. I would also like to thank the remainder of my committee, Barb Butler and Trevor Charles, for their opinions and insight. Thank you to the Neufeld lab members for their continued input and for making the lab such an enjoyable place to spend the last two years. I would like to specifically thank Katja Engel for her continued input and help with troubleshooting throughout this project, and Daniel Min and Jackson Tsuji for technical support with QIIME2, R, and Python. Thank you to the people who collected the clay and rock samples, without which this research would not have been possible, and to the Nuclear Waste Management Organization, Ontario Research Fund, and the Natural Sciences and Engineering Research Council for funding this research. Finally, I would like to thank my family and friends for their ongoing support. vi Table of Contents Author's Declaration ................................................................................................................. ii Statement of Contributions ......................................................................................................iii Abstract.................................................................................................................................... iv Acknowledgements.................................................................................................................. vi List of Figures.......................................................................................................................... ix List of Tables ........................................................................................................................... xi List of Abbreviations .............................................................................................................. xii Chapter 1 Introduction and literature review............................................................................ 1 1.1 Canadian used nuclear fuel ............................................................................................. 1 1.2 Storage of used nuclear fuel............................................................................................ 2 1.3 Bentonite clay.................................................................................................................. 3 1.4 Environmental conditions of a DGR............................................................................... 4 1.5 Importance of studying DGR microbiology.................................................................... 6 1.6 Biogeochemical cycling relevant to a DGR.................................................................... 6 1.6.1 Primary Production in a DGR environment ............................................................. 7 1.6.2 Chemoorganoheterotrophic metabolisms................................................................. 9 1.6.3 Sulfide sequestration............................................................................................... 10 1.7 Preventing microbial growth in a DGR......................................................................... 12 1.8 Methods for characterizing the microbiology of bentonite clay ................................... 13 1.9 Objectives...................................................................................................................... 14 1.9.1 Sample sites............................................................................................................ 15 Chapter 2 Materials and Methods........................................................................................... 16 2.1 Sampling and storage .................................................................................................... 16 2.2 Sample preparation for DNA extraction, cultivation, water activity, and moisture content................................................................................................................................. 16 2.2.1 Tsukinuno bentonite and Opalinus clay samples ................................................... 17 2.2.2 Northern Ontario Crystalline rock samples............................................................ 17 2.3 Water activity and moisture content.............................................................................. 17 vii 2.4 Cultivation..................................................................................................................... 17 2.4.1 Aerobic and anaerobic heterotrophs....................................................................... 18 2.4.2 Sulfate reducing bacteria ........................................................................................ 18 2.5 DNA extraction ............................................................................................................. 18 2.5.1 Clay and rock samples............................................................................................ 19 2.5.2 Cultures................................................................................................................... 19 2.5.3 Swabs...................................................................................................................... 19 2.6 DNA quantification....................................................................................................... 20 2.7 Quantitative PCR........................................................................................................... 20 2.8 Amplification and sequencing of 16S rRNA genes ...................................................... 21 2.9 Sequence analysis.......................................................................................................... 22 Chapter 3 Results and discussion............................................................................................ 24 3.1 Description of samples.................................................................................................. 24 3.2 Microbial abundance..................................................................................................... 30 3.3 Microbial viability......................................................................................................... 35 3.4 Comparison of clay and rock reads to control reads ..................................................... 36 3.5 Comparison of Tsukinuno bentonite samples............................................................... 37 3.6 Diversity of clay and rock samples ............................................................................... 41 3.7 Microbial characterization of clay and rock samples.................................................... 44 3.7.1 Tsukinuno bentonite samples ................................................................................. 44 3.7.2 Tsukinuno bentonite-associated cultures................................................................ 53 3.7.3 Opalinus clay and NOCR samples ......................................................................... 55 3.8 Predicting consistency of 16S rRNA gene profiles before sequencing ........................ 58 3.9 Heterogeneity of ancient natural barrier component profiles........................................ 59 Chapter 4 Conclusions and recommendations........................................................................ 60 Bibliography ........................................................................................................................... 64 Appendix A Supplemental figures.......................................................................................... 84 viii List of Figures Figure 1.1 Nuclear fuel pellets stacked to form “pencils” that are welded into fuel bundles [2].............................................................................................................................................. 1 Figure 1.2 Radioactivity of irradiated uranium over time compared to the radioactivity of uranium ore [1]. ........................................................................................................................ 2 Figure 1.3 Theoretical design of a deep geological repository. Modified from [10, 11]......... 3 Figure 3.1 Images of the ten Tsukinuno bentonite samples immediately after arrival at the University of Waterloo. .......................................................................................................... 26 Figure 3.2 Temperature inside the package containing the Tsukinuno bentonite samples between the time of sampling and arrival at the University of Waterloo. .............................. 27 Figure 3.3 Water activity and moisture content of the Tsukinuno bentonite samples........... 28 Figure 3.4 Opalinus clay core (O1), and NOCR cores N2 (99.0-99.3 m belowground), N4 (219.7-219.9 m belowground), and N6 (513.4-513.7 m belowground). ................................ 29 Figure 3.5 Abundance of 16S rRNA gene copies, cultivatable aerobic heterotrophs, anaerobic heterotrophs, and SRB in the Tsukinuno bentonite samples from Japan............... 31 Figure 3.6 Number of 16S rRNA gene copies per gram of clay in the Tsukinuno bentonite samples from Japan, the NOCR samples, and the Opalinus clay sample............................... 33 Figure 3.7 Scatterplots showing the relationship between the number of 16S rRNA gene copies/gdw and the moisture content (A) or water activity (B) of the Tsukinuno bentonite samples.................................................................................................................................... 34 Figure 3.8 Violin plots of the distribution of sequenced reads for the Tsukinuno bentonite, Opalinus clay, and NOCR samples and their corresponding controls.................................... 37 Figure 3.9 Principal coordinate analysis (PCoA) plots of Bray Curtis distances (A), Jaccard distances (B), weighted UniFrac distances (C), and unweighted UniFrac distances (D) of the Tsukinuno bentonite samples.................................................................................................. 39 Figure 3.10 PCoA triplot based on Bray Curtis distances of the Tsukinuno bentonite samples.................................................................................................................................... 40 ix Figure 3.11 A measure of total diversity (Shannon index) compared to richness (Faith’s phylogenetic diversity; A) and to evenness (Pielou’s evenness, B) in the Tsukinuno bentonite, Opalinus clay, and NOCR samples........................................................................ 42 Figure 3.12 Violin plots showing the Shannon diversity of the aerobic heterotrophs (AH), anaerobic heterotrophs (AnH), and sulfate reducing bacteria (SRB), as well as the Tsukinuno bentonite samples, the Opalinus clay sample, and the NOCR samples.................................. 43 Figure 3.13 Bubble plot showing the 16S rRNA gene profiles of the Tsukinuno bentonite clay samples (blue) and the corresponding aerobic heterotroph (AH), anaerobic heterotroph (AnH), and SRB cultures (grey). ............................................................................................ 45 Figure 3.14 Bubble plot showing the relative abundance of ASVs predicted to have the listed functions in the Tsukinuno bentonite samples and the corresponding aerobic heterotroph (AH), anaerobic heterotroph (AnH) and SRB cultures........................................................... 48 Figure 3.15 Bubble plot showing the 16S rRNA gene profile of the Opalinus clay sample. 57 Figure 3.16 Bubble plot showing the 16S rRNA gene profile of the inner and swabbed outer layer of the three NOCR samples. Two biological replicates of each rock sample are included. Only ASVs at or above 5% relative abundance in a sample are included. ............. 58 Figure S1 Histograms showing the distribution of score statistics assigned by Decontam to the ASVs detected in the clay and rock sample DNA amplified with 40 (A), 45 (B), and 50 (C) cycles of PCR. .................................................................................................................. 84 Figure S2 Bubble plot showing the 16S rRNA gene profiles of the ten Tsukinuno bentonite clay samples (blue) and the corresponding aerobic heterotroph (AH), anaerobic heterotroph (AnH), and SRB cultures (grey), with ASVs removed by Decontam with a theshold value of 0.5 indicated (red outline)....................................................................................................... 85 Figure S3 Bubble plot showing the 16S rRNA gene profiles of the Opalinus clay samples, with ASVs removed by Decontam with a threshold value of 0.5 indicated (red outline). ..... 86 Figure S4 Bubble plot showing the 16S rRNA gene profiles of the NOCR samples, with ASVs removed by Decontam with a threshold value of 0.5 indicated (red outline). ............. 87 xList of Tables Table 3.1 List of Tsukinuno bentonite samples and site characteristics. ............................... 25 xi List of Abbreviations <ul style="display: flex;"><li style="flex:1">Acetyl-coenzyme A </li><li style="flex:1">Acetyl-CoA </li></ul>ANME AOM APM ASV ATP BSA CANDU CFU CH4 Anaerobic methanotrophic Anaerobic oxidation of methane Adaptive phased management Amplicon sequence variant Adenosine triphosphate Bovine serum albumin CANada Deuterium Uranium Colony forming units Methane <ul style="display: flex;"><li style="flex:1">Cl- </li><li style="flex:1">Chloride </li></ul><ul style="display: flex;"><li style="flex:1">CO2 </li><li style="flex:1">Carbon dioxide </li></ul>DGR Fe2+ Deep geological repository Iron(II) Fe3+ Iron(III) <ul style="display: flex;"><li style="flex:1">gdw </li><li style="flex:1">Gram dry weight </li></ul>H2 Dihydrogen <ul style="display: flex;"><li style="flex:1">HS- </li><li style="flex:1">Bisulfide </li></ul>MAG MIC MPN N2 Metagenome assembled genome Microbiologically influenced corrosion Most probable number Dinitrogen +<ul style="display: flex;"><li style="flex:1">NH4 </li><li style="flex:1">Ammonium </li></ul>-<ul style="display: flex;"><li style="flex:1">NO2 </li><li style="flex:1">Nitrite </li></ul>-<ul style="display: flex;"><li style="flex:1">NO3 </li><li style="flex:1">Nitrate </li></ul>NOCR NWMO OTU Northern Ontario crystalline rock Nuclear Waste Management Organization Operational taxonomic unit xii PCR PLFA qPCR QIIME2 rPolymerase chain reaction Phospholipid fatty acid Quantitative polymerase chain reaction Quantitative Insights into Microbial Ecology 2 Pearson correlation Spearman correlation Reasoner’s 2A rs R2A rRNA S0 Ribosomal RNA Elemental sulfur S2- Sulfide 2- S2O3 Thiosulfate 2- <ul style="display: flex;"><li style="flex:1">SO4 </li><li style="flex:1">Sulfate </li></ul>SRB TCA UFC Sulfate reducing bacteria Tricarboxylic acid Used fuel container xiii Chapter 1 Introduction and literature review 1.1 Canadian used nuclear fuel Canadian nuclear reactors, also known as CANDU reactors, use solid uranium dioxide as fuel. Uranium dioxide powder is pressed to form small fuel pellets, which are stacked together and housed in a zirconium alloy casing to form fuel pencils that are then welded together within bundles, which is the “battery” that fuels a nuclear reactor (Figure 1.1) [2]. After a fuel bundle has served its purpose in a nuclear reactor and is removed, it remains radioactive and must be stored safely for approximately one million years until it returns to the radioactivity level of naturally occurring uranium (Figure 1.2) [4]. Considering both the existing used nuclear fuel and an additional 90,000 used fuel bundles that are produced every year from use in Canadian reactors, projections suggest that 5.5 million used fuel bundles must be stored by the end of current Canadian nuclear reactor lifetimes [5].

Microbiology of Barrier Component Analogues of a Deep Geological Repository

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